Matricellular proteins are thought to function mainly on the cell surface to transduce signals between the extracellular and intracellular environment, regulating cell proliferation, migration and survival. The most severe chondrodysplasia described to date for loss of a matricellular protein occurs when CCN2 is absent. Upon closer inspection of the Ccn2 phenotype, major defects in extracellular matrix (ECM) secretion/assembly were observed. In particular, increased ER stress was observed in Ccn2 mutant chondrocytes. This result was unexpected because the Ccn2 mutant allele is a true null allele-no mRNA or protein is made, and all other mutations in matrix proteins described to date that lead to ER stress are caused by expression of a truncated or misfolded gene product. Conversely, an increase in CCN2 levels, achieved through transgenic overexpression in the growth plate, resulted in attenuation of ER stress. Moreover, Ccn2 mRNA expression was found to be activated by ER stress with the same kinetics observed for classic primary ER stress response genes, whereas the expression of mRNAs encoding cartilage ECM proteins decreases during ER stress. The novel ER stress phenotype will be characterized in Aim 1. The observation that the majority of CCN2 exhibits an intracellular localization suggests that CCN2 may play a protective, "chaperone-like" role in the secretory pathway. This possibility will be investigated in Aim 2. These findings thus may challenge the paradigm that matricellular proteins such as CCNs function solely on the cell surface. Finally, CCN proteins interact with integrins, and it is known that integrin signaling impacts both reactive oxygen species (ROS) and hypoxic signaling. We hypothesize that if CCN2 acts extracellularly to protect against ER stress, it does so through effects on ROS or hypoxia pathways. Aim 3 will test this hypothesis. Hence, the specific hypotheses to be tested in this proposal are (1) that loss of CCN2 exacerbates ER stress and conversely, that overexpression of CCN2 is protective against ER stress, (2) CCN2 acts as an intracellular "scaffold" or "chaperone" to promote correct assembly of ECM protein complexes, and (3) that CCN2 may protect against ER stress through either ROS- or hypoxia-mediated mechanisms. PUBLIC HEALTH RELEVANCE: Research is increasingly focusing on the regulation of matrix synthesis and degradation, as these are hallmarks of healthy and diseased cartilage. Moreover, the majority of human chondrodysplasias are caused by abnormal retention of mis-folded ECM proteins, which results in endoplasmic reticulum (ER) stress leading to irreversible changes in cell viability and matrix secretion. Hence, understanding the regulatory mechanisms of ECM protein processing and secretion in cartilage is essential to the development of future therapies and drug targets for cartilage maintenance, repair and regeneration.